Patients with Crohn's disease (CD) have a defective intestinal epithelial tight junction (TJ) barrier manifested by an increase in intestinal permeability. The defective intestinal epithelial TJ barrier appears to be an important pathogenic factor of CD, which allows intestinal penetration of toxic luminal antigens and substances leading to the intestinal inflammation. Tumor necrosis factor-alpha (TNF-alpha) plays a central causative role in intestinal inflammation of CD. Several recent studies including our preliminary studies demonstrated that TNF-alpha produces a persistent increase in intestinal epithelial TJ permeability. The TNF-alpha induced increase in intestinal epithelial TJ permeability could be an important pro-inflammatory mechanism, which allows increased intestinal permeation of toxic luminal antigens. Since TNF-alpha plays a central role in the intestinal inflammation of CD, understanding the intracellular mechanisms involved in TNF-( induced increase in intestinal TJ permeability will be crucial in developing potential therapeutic strategies to prevent the abnormal increase in intestinal TJ permeability. In this grant application, we propose to delineate the cellular and molecular mechanisms, which mediate the TNF-alpha induced increase in intestinal TJ permeability, using the Caco-2 Intestinal epithelial cells. Based on our preliminary data, we hypothesize that TNF-alpha induced NF-kappaB activation is a key intracellular process, which regulates the TNF-alpha modulation of the intestinal epithelial TJ barrier. The proposed specific aims of this grant application will test the hypothesis that NF-(B activation is a key intracellular process regulating the TNF-alpha induced increase in intestinal epithelial TJ (or paracellular) permeability. The proposed specific aims will also 1) delineate the intracellular mechanisms which regulate the TNF-alpha induced NF-kappaB activation and increase in intestinal epithelial TJ permeability, 2) determine the molecular and cellular mechanisms by which TNF-alpha regulates the TJ proteins, and 3) determine the possible intracellular targets for therapeutic intervention to prevent the TNF-alpha induced increase in intestinal TJ permeability.